Natural killer (NK) cells are a subset of spontaneously cytotoxic lymphocytes that lytically destroy tumor cells without apparent antigen specificity or restriction by histocompatibility molecules. Lymphokines are lymphocyte-derived peptides that modulate immunologic and inflammatory responses by regulating the activity, growth and differentiation of a wide variety of leukocyte and nonleukocyte target cells. Similar factors produced by a variety of cell types, together with lymphokines, are known as cytokines. Several cytokines are known to stimulate proliferation of NK cells and to enhance their cytotoxic activity.
Interleukin-2 (IL-2), formerly T-cell growth factor (TCGF), is a T-cell-derived cytokine. Since 1985, IL-:2 has been used in the treatment of human neoplasia, mainly in patients with metastasizing solid tumors, such as malignant melanoma and renal cell carcinoma (Rosenberg et al., N. Engl. J. Med. 316:889-897 (1987); Bukowski et al., J. Clin. Oncol. 7:477-485 (1989)), but more recently also in acute myelogenous leukemia (AML) (Foa et al., Br. J. Haematol. 77:491-496.3 (1991)). In the initial studies, IL-2 was administered together with autologous lymphocytes that had been treated with IL-2 in vitro, but in recent years IL-2 has more frequently been administered as a single agent.
The high expectations for the treatment of human cancer using IL-2 were based on the findings that treatment with IL-2 can induce the regression of established tumors in several animal tumor models in vivo (Rosenberg et al., J. Exp. Med. 161:1169-1188 (1985); Lotre and Rosenberg, in Interleukin-2, K. A. Smith, ed., Academic Press, San Diego, pp. 237-294 (1988)). The mechanism underlying this anti-tumor effect of IL-2 has been much debated, but accumulating evidence points to the anti-tumor effector cell as the natural killer (NK)-cell. Depletion of NK cells from experimental animals eliminates the anti-tumor effect of IL-2 in many experimental models for tumor growth and metastasis (Mule et al., J. Immunol. 139:285 (1987)). Further, the only subset of resting human peripheral blood lymphocytes that carry transducing receptors for IL-2 (IL-2R) on the cell surface are NK cells (Caliguri et al., J. Clin. Invest. 91:123-132 (1993)).
IL-2 activates many NK-cell functions, including baseline or "natural" anti-tumor cytotoxicity, antibody-dependent cellular cytotoxicity (ADCC), proliferation, and cytokine production (Trinchieri, Adv. Immumol. 47:187-376 (1989)). Also, IL-2-activated NK cells, frequently referred to as lymphokine-activated killer (LAK) cells, display a broader spectrum of reactivity against human and murine tumor target cells. Thus, NK cells activated by IL-2 not only kill NK cell-sensitive tumor cells more efficiently, but also kill tumor cells that are insensitive to the constitutive cytotoxic activity mediated by NK cells.
Recent studies have also shown that IL-2, when combined with histamine or serotonin, augments NK cell cytotoxicity in the presence of monocytes in vitro (Hellstrand et al., J. Immunol. 145(12):4365-4370 (1990) and Hellstrand et al., Scand. J. Immunol. 32(2) :183-192 (1990)). These studies suggest an interaction between monocytes and NK cells that is subject to regulation by these biogenic amines (Hellstrand et al., J. Interferon Rsch. 12:199-206 (1992). These NK cell regulating mechanisms are thus believed to be of importance to the NK cell mediated response to metastatic tumors in vivo.
Despite the beneficial effects obtained with IL-2 therapy in experimental animals and despite the remarkable effects of IL-2 on the killing activity of human NK cells in vitro, the results of the clinical trials of IL-2 in human cancer have, as yet, been disappointing. Only a small fraction of patients with metastatic melanoma or renal cell carcinoma show objective regression of tumor burden after treatment with very high doses of IL-2 (Bukowski et al., J. Clin. Oncol. 7:477-485 (1989); Whitehead et al., J. Natl. Cancer Inst. 83:1250-1253 (1991)). In addition, IL-2 produces severe side effects, including hypotension, fluid retention ("capillary leak syndrome"), fever, lethargy and nausea.
Other interleukins are also known to stimulate NK cell activity. For example, IL-12, also known as natural killer cell stimulatory factor (NKSF), is a recently discovered cytokine which has also been reported to increase NK cell and cytotoxic T lymphocyte activity, T cell proliferation, and the production of interferon-T. It has been found to enhance the spontaneous cytotoxic activity of peripheral blood lymphocytes against a variety of tumor-derived target cell lines (Chehimi et al., J Exp. Med. 175:789-796 (1992)). IL-1 is another cytokine known to enhance NK cell cytotoxicity.
The interferons consist of a family of secreted proteins with potent antiproliferative and immunomodulatory activities. These immunomodulatory effects include activation of macrophages, augmentation of cellular and humoral immune responses, and enhancement of NK-cell activity. All three major subtypes of human interferon, i.e., interferon-a (IFN-.alpha.), interferon-.beta. (IFN-.beta.) and interferon-.gamma. (IFN-.gamma.), are known to enhance NK cell cytotoxicity. Interferon-.alpha. (IFN-.alpha.) is a major regulatory factor for NK cells. It has been found to stimulate NK cells (Silva et al., J. Immunol. 125:479-484 (1980)) and augment NK cell cytotoxicity both in vitro and in vivo (Trinchieri, Adv. Immunol. 47:187-376 (1989)). Although IFN-.alpha. has been shown to be effective with some neoplasias, the overall results of therapy with high doses of IFN-.alpha. have been disappointing. In addition, patients treated with IFN-.alpha. often have acute toxic reactions including fever, chills, myalgias, anorexia, fatigue, headache, nausea and vomiting.
Other known stimulators of NK cell activity include certain flavonoids. The flavonoids are a group of low molecular weight polyphenolic secondary plant metabolites. Flavone-8-acetic acid has been found to potently augment NK activity in the spleen, liver, lungs, and peritoneum (Wiltrout et al., J. Immunol. 140(9):3261-3265 (1988). Xanthenone-4-acetic acid (XAA), an analog of FAA, and its methyl-substituted derivatives, have also been found to induce NK activity in vitro (Ching et al., Eur. J. Cancer 27(1):79-83 (1991)). Clinical trials of FAA have been disappointing, however, due to non-linear pharmokinetics, low dose potency and problems of drug precipitation.